The goal of the proposed research is to understand the role of molecular dynamics in the function of certain energy-transducing membranes. The three membrane proteins on which the research will focus are: (1) the Ca 2 ion minus ATPase of rabbit skeletal muscle sarcoplasmic reticulum, (2) rhodopsin from the disc membrane of the bovine retinal rod, (3) cytochrome c oxidase from the inner membrane of the bovine cardiac mitochondrion. During its initial stages, the project will be concerned primarily with the Ca 2 ion minus ATPase. The rotational and translational motions of proteins and lipids, and their spatial arrangements will be investigated with spectroscopic techniques: (1) Electron paramagnetic resonance (EPR, both convential and saturation transfer methods) will be used to detect the time-averaged rotational motion of spin-labeled proteins and lipids. (2) A novel pulsed laser spectrometer will be constructed and used to obtain complementary time-resolved information on rotational motion, probing either extrinsic fluorescent and phosphorescent labels or intrinsic chromophores. (3) The pulsed laser system will be used to study the spatial arrangement of molecules and their translational diffusion, using time-resolved fluorescence energy transfer measurements and lanthanide ions (terbium and europium). By studying a single system with several techniques, ambiguities in interpretation will be minimized. By performing similar experiments on three different systems, opportunities for comparison will be maximized. The use of spectroscopic probes will permit observations during membrane function, indicating which motions may be coupled to function. Experiments will be performed, whenever possible, on native membranes as well as on reconstituted membranes, in which the composition can be varied. Perturbations in lipid composition, protein composition, temperature, and other physiologically relevant parameters will be applied. The resulting effects on lipid mobility, protein mobility, and biochemical activity will be analyzed to further elucidate the features of molecular motion and interaction that affect the activities of these systems.